Department of Nephrology and Hypertension, University Medical Center Utrecht, University of Utrecht, The Netherlands.

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Department of Pathology, Ann and Robert H. Lurie Children's Hospital of Chicago, Northwestern University's Feinberg School of Medicine and Robert H. Lurie Cancer Center, IL, USA.

Abstract

We recently identified hypermethylation at the gene promoter of transcription factor 21 (TCF21) in clear cell sarcoma of the kidney (CCSK), a rare pediatric renaltumor. TCF21 is a transcription factor involved in tubular epithelial development of the kidney and is a candidate tumor suppressor. As there are no in vitro models of CCSK, we employed a well-established clear cellrenalcell carcinoma (ccRCC) cell line, 786-O, which also manifests high methylation at the TCF21 promoter, with consequent low TCF21 expression. The tumor suppressor function of TCF21 has not been functionally addressed in ccRCC cells; we aimed to explore the functional potential of TCF21 expression in ccRCC cells in vitro. 786-O clones stably transfected with either pBABE-TCF21-HA construct or pBABE vector alone were functionally analyzed. We found that ectopic expression of TCF21 in 786-O cells results in a trend toward decreased cellproliferation (not significant) and significantly decreased migration compared with mock-transfected 786-O cells. Although the number of colonies established in colony formation assays was not different between 786-O clones, colony size was significantly reduced in 786-O cells expressing TCF21. To investigate whether the changes in migration were due to epithelial-to-mesenchymal transition changes, we interrogated the expression of selected epithelial and mesenchymal markers. Although we observed upregulation of mRNA and protein levels of epithelial marker E-cadherin in clones overexpressing TCF21, this did not result in surface expression of E-cadherin as measured by fluorescence-activated cell sorting and immunofluorescence. Furthermore, mRNA expression of the mesenchymal markers vimentin (VIM) and SNAI1 was not significantly decreased in TCF21-expressing 786-O cells, while protein levels of VIM were markedly decreased. We conclude that re-expression of TCF21 in renal cancer cells that have silenced their endogenous TCF21 locus through hypermethylation results in reduced clonogenicproliferation, reduced migration, and reduced mesenchymal-like characteristics, suggesting a tumor suppressor function for transcription factor 21.

TCF21 methylation, expression, and reconstitution. (A) Schematic of the TCF21 gene, showing relative position of the CpG islands to the first three exons. (B) TCF21 mRNA expression levels in 786‐pBABE‐mock and 786‐O pBABE‐TCF21 cells, as measured by qPCR. Data are presented as mean ± SEM. N = 2 independent experiments, each performed in triplicate. (C) Western blots of eight clones of 786‐O that were used for this study; four clones ‘(−)’ were mock‐transfected with pBABE‐puro (N1F4, B5F1, B6D10, and N1G4) and four ‘(+)’ were transfected with HA‐tagged pBABE‐TCF21 (2B12, 5D2, 9D12, and 9H9). Upper blot was immunostained with anti‐HA to detect exogenous TCF21 (18 kDa), while lower blot is loading control stained with anti‐actin (42 kDa). N = 2 independent experiments, representative result shown. (D) Treating 786‐O cells with 3 μm decitabine increases the expression of TCF21 mRNA, as measured by qPCR. N = 1, performed in duplicate. *P < 0.05.

TCF21 expression reduces migration of cells in vitro. (A) Representative photographs of DAPI‐stained nuclei after migration through a Boyden chamber of 786‐O pBABE‐mock cells and 786‐O pBABE‐TCF21 cells. (B) Sample curve of impedance (cell index) from a representative experiment with all clones over 24 h, from which slopes were calculated. (C) The rate of change in impedance of cells across a transwell membrane in an XCeLLigence system indicates that clones expressing TCF21 migrate significantly slower than pBABE‐mock‐transfected cells. Data are presented as mean ± SEM. N = 3 independent experiments; each experiment had four technical replicates. *P < 0.05.

Mesenchymal marker VIM is downregulated by TCF21. Three clones expressing pBABE‐TCF21 show reduced levels of VIM on western blot as compared to four pBABE‐mock clones. Clone 9D12 was not included in the analysis as it did not express TCF21 at the time lysates were prepared (Fig. B). N = 2 independent experiments.